This invention relates to a series of tetrahydro- and dihydroquinoline, tetrahydronaphthalene and tetrahydro-5H-benzocycloheptene bicyclic compounds of Formulae (I) and (II) and non-toxic salts thereof, which selectively antagonize the xcex1vxcex23 integrin while minimally inhibiting platelet aggregation mediated by xcex1IIbxcex23 integrin and are useful as bone antiresorptive agents.
The present invention relates to fused bicyclic derivatives which exhibit activity as bone antiresorptive agents by inhibition of the osteoclast vitronectin receptor(xcex1vxcex23). The integrin xcex1vxcex23 has been shown to mediate the invasion of cancerous melanoma cells into healthy tissue (Seftor et al., Proc. Natl. Acad. Sci, USA, 1992, 89, 1557-1561) and to protect these cells against natural cell death cycle (apoptosis) (Montgomery et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 8856-8860). Vitronectin receptor (xcex1vxcex23) antagonists have been shown to inhibit the growth of various solid tumors of human origin (Brooks et al., Cell, 1994, 79, 1157-1164). More recently, xcex1vxcex23 has been shown to be involved in liver metastasis (Yun et al., Cancer Res., 1996, 56, 3103-3111). Although angiogenesis is an important and natural process in growth and wound healing, it is now appreciated that a variety of clinically relevent conditions are pathologically related to these processes, and that the integrin xcex1vxcex23 is involved. For example, xcex1vxcex23 was shown to be expressed on human wound tissue but not on normal skin (Brooks, et al., Science, 1994, 264, 569-571) and is preferentially expressed on angiogenic blood vessels, such as those feeding a growing/invading tumor. It has also been shown that antagonists of xcex1vxcex23 promote tumor regression by inducing apoptosis of the tumor cells (Brooks et al., Cell, 1994, 79, 1157-1164). The process of neovascularization (new blood vessel growth, angiogenesis), which is critical for tumor growth and metastasis, is also an important event in occular tissue, leading to diabetic retinopathy, glaucoma and blindness (Adamis et al., Am. J. Ophthal., 118, 445-450(1994); Hammes et al., Nature Med., 1996, 2,529-533; Friedlander, et al., Natl. Acad. Sci. U.S.A., 1996, 93, 9764-9769) and in joints, promoting rheumatoid arthritis (Peacock et al., J. Exp. Med., 1992, 175, 1135-1138). xcex1vxcex23 has been shown to play a pivotal role in the proliferation and migration of smooth muscle and vascular endothetial cells, a pathological process leading to restenosis after balloon angioplasty (Choi et al., J. Vasc. Surgery, 1994, 19, 125-134; Matsuno et al., Circulation, 1994, 90, 2203-2206). At least one type of virus (adenovirus) has been shown to utilizeo xcex1vxcex23 for entering host cells (White et al., Current Biology, 1993, 596-599).
Various bone diseases involve bone resorption, the dissolution of bone matter, which is mediated by only one known class of cells, the osteoclasts. When activated for resorption, these motile cells initially bind to bone, a process well known to be mediated by xcex1vxcex23 (Davies et al., J. Cell. Biol., 1989 109, 1817-1826; Helfrich et al., J Bone Mineral Res., 1992, 7, 335-343). It is also well known that blockade of xcex1vxcex23 with antibodies or peptides containing the sequence arginine-glycine-aspartic acid (RGD) blocks osteoclast cell adhesion and bone resorption in vitro (Horton et al., Exp. Cell Res. 1991, 195, 368-375) and that echistatin, an RGD containing protein, inhibits bone resorption in vivo (Fisher et al., Endocrinolog. y, 1993, 132, 1411-1413). More recently, an RGD peptidomimetic has likewise been shown to inhibit osteoclasts in vitro and, by iv administration prevents osteoporosis in vivo (Engleman et al., J. Clin. Invest., 1997, 99, 2284-2292).
A series of bicyclic compounds having a nucleus formed of two fused six-membered rings which include isoquinoline, isoquinolone, tetrahydronaphthalene, dihydronaphthalene or tetralone substituted with both basic and acidic functionality and which are useful in inhibition of platelet aggregation are disclosed in EP 0635492, WO96/22288, U.S. Pat Nos. 5,618,843 and 5,731,324 and are described by Formula I 
The current major bone diseases of public concern are osteoporosis, hypercalcemia of malignancy, osteopenia due to bone metastases, periodontal disease, hyperparathyroidism, periarticular erosions in rheumatoid arthritis, Paget""s disease, immobilization-induced osteopenia and the result of glucocorticoid treatment.
All these conditions are characterized by bone loss, resulting from an imbalance between bone resorption (breakdown) and bone formation, which continues throughout life at the rate of about 14% per year on the average. However, the rate of bone turnover differs from site to site, for example, it is higher in the trabecular bone of the vertebrae and the alveolar bone in the jaws than in the cortices of the long bones. The potential for bone loss is directly related to turnover and can amount to over 5% per year in vertebrae immediately following menopause, a condition which leads to increased fracture risk.
There are currently 20 million people with detectable fractures of the vertebrae due to osteoporosis in the United States. In addition, there are 250,000 hip fractures per year attributed to osteoporosis. This clinical situation is associated with a 12% mortality rate within the first two years, while 30% of the patients require nursing home care after the fracture.
The minimal inhibition of platelet aggregation mediated by xcex1IIbxcex23 integrin while selectively antagonizing the xcex1vxcex23 integrin and thus being available as bone antiresorptive agents is an important benefit of compounds of the invention and is important in mammals, especially man.
Accordingly, the present invention discloses bicyclic compounds represented by general Formula (I): 
wherein:
xe2x80x94 xe2x80x94 xe2x80x94 represents the presence of an optional double bond;
n is an integer of 2 to 5;
v is an integer of 0 or 1;
Axe2x80x94B is a diradical of the formulae: 
m is an integer of 1 or 2;
Y is selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94, 
R1 is hydrogen or straight chain alkyl of 1 to 6 carbon atoms; phenylalkyl wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the phenyl moiety is optionally substituted with one or more substituents which may be the same or different and are selected from hydroxy, amino, halogen, straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, cyano, nitro, alkylamino of 1 to 6 carbon atoms, and dialkylamino of 1 to 6 carbon atoms; heterocyclylalkyl, wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the heterocyclyl moiety is selected from a 5- or 6-membered heterocyclic ring which contains 1 to 3 heteroatoms which may be the same or different, selected from nitrogen, oxygen and sulfur optionally substituted with one or more substituents which may be the same or different, and are selected from hydroxy, amino, halogen, straight chain alkyl of 1 to 6 carbon atoms, cyano and nitro;
R1a is hydrogen or straight chain alkyl of 1 to 6 carbon atoms; phenylalkyl wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the phenyl moiety is optionally substituted with one or more substituents which may be the same or different and are selected from hydroxy, amino, halogen, straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, cyano, nitro, alkylamino of 1 to 6 carbon atoms, and dialkylamino of 1 to 6 carbon atoms;
R2 is hydrogen, xe2x80x94NHR1, or xe2x80x94OR1; aryl of 6 to 12 carbon atoms optionally substituted with one or more substituents selected from straight chain alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms, xe2x80x94S-alkyl of 1 to 6 carbon atoms, cyano, nitro, halogen and phenyl; the heterocyclyl moiety is selected from a 5- or 6-membered heterocyclic ring which contains 1 to 3 heteroatoms which may be the same or different,selected from nitrogen, oxygen and sulfur optionally substituted with one or more substituents which may be the same or different, and are selected from hydroxy, amino, halogen, straight chain alkyl of 1 to 6 carbon atoms, cyano and nitro; phenylalkyl wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the phenyl moiety is optionally substituted with one or more substituents which may be the same or different and are selected from hydroxy, amino, halogen, straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, cyano, nitro, alkylamino of 1 to 6 carbon atoms, and dialkylamino of 1 to 6 carbon atoms; heterocyclylalkyl, wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the heterocyclyl moiety is selected from a 5- or 6-membered heterocyclic ring which contains 1 to 3 heteroatoms which may be the same or different,selected from nitrogen, oxygen and sulfur optionally substituted with one or more substituents which may be the same or different, and are selected from hydroxy, amino, halogen, straight chain alkyl of 1 to 6 carbon atoms, cyano and nitro;
G is a moiety selected from the group consisting of: 
u is an integer of 0 or 1;
R4 is straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, alkoxy, or phenylalkyloxy wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the phenyl moiety is optionally substituted with one or more substituents which may be the same or different and are selected from hydroxy, amino, halogen, straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, cyano, nitro, alkylamino of 1 to 6 carbon atoms, and dialkylamino of 1 to 6 carbon atoms;
R5 is hydrogen, straight chain alkyl of 1 to 6 carbon atoms, or phenylalkyl wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the phenyl moiety is optionally substituted with one or more substituents which may be the same or different and are selected from hydroxy, amino, halogen, straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, cyano, nitro, alkylamino of 1 to 6 carbon atoms, and dialkylamino of 1 to 6 carbon atoms;
R5a is hydrogen, straight chain alkyl of 1 to 6 carbon atoms, or phenylalkyl wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the phenyl moiety is optionally substituted with one or more substituents which may be the same or different and are selected from hydroxy, amino, halogen, straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, cyano, nitro, alkylamino of 1 to 6 carbon atoms, and dialkylamino of 1 to 6 carbon atoms;
R5b is hydrogen, straight chain alkyl of 1 to 6 carbon atoms, or phenylalkyl wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the phenyl moiety is optionally substituted with one or more substituents which may be the same or different and are selected from hydroxy, amino, halogen, straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, cyano, nitro, alkylamino of 1 to 6 carbon atoms, and dialkylamino of 1 to 6 carbon atoms;
provided that the optional double bond xe2x80x94 xe2x80x94 xe2x80x94 is a single bond when Axe2x80x94B is the diradical xe2x80x94CH2xe2x80x94(CH2)mxe2x80x94;
or a pharmaceutically acceptable salt thereof.
Among the preferred groups of compounds of Formula (I) of this invention including pharmaceutically acceptable salts thereof are those in the subgroups wherein:
a)
n is an integer of 2 to 4;
the moiety 
is located at the a or b position of the bicyclic nucleus;
R1 is hydrogen or straight chain alkyl of 1 to 6 carbon atoms; phenylalkyl wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the phenyl moiety is optionally substituted with one or two substituents which may be the same or different and are selected from halogen, straight chain alkyl of 1 to 6 carbon atoms, and nitro; heterocyclylalkyl, wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the heterocyclyl moiety is selected from 2- or 3-furyl, 2- or 3-thienyl, and 2-, 3- or 4-pyridyl optionally substituted with one or two, substituents which may be the same or different, and are selected from halogen, straight chain alkyl of 1 to 6 carbon atoms, and nitro;
R2 is hydrogen; aryl of 6 to 12 carbon atoms optionally substituted with one or more substituents selected from straight chain alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms, nitro, and halogen; the heterocyclyl moiety is selected from 2- or 3-furyl, 2- or 3-thienyl, and 2-, 3- or 4-pyridyl; phenylalkyl wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the phenyl moiety is optionally substituted with one or more substituents which may be the same or different and are selected from halogen, straight chain alkyl of 1 to 6 carbon atoms, and nitro; heterocyclylalkyl, wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the heterocyclyl moiety is selected from 2- or 3-furyl, 2- or 3-thienyl, and 2-, 3- or 4-pyridyl;
the optional double bond xe2x80x94 xe2x80x94 xe2x80x94 is a single bond;
where m, u, v, G, Y, Axe2x80x94B, R1a, R4, R5a, and R5b are hereinbefore defined;
b)
n is an integer of 2 to 4;
the moiety 
is located at the a or b position of the bicyclic nucleus; Axe2x80x94B is the diradical xe2x80x94CH2xe2x80x94(CH2)mxe2x80x94;
R1 is hydrogen or straight chain alkyl of 1 to 6 carbon atoms; phenylalkyl wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the phenyl moiety is optionally substituted with one or two substituents which may be the same or different and are selected from halogen, straight chain alkyl of 1 to 6 carbon atoms, and nitro; heterocyclylalkyl, wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the heterocyclyl moiety is selected from 2- or 3-furyl, 2- or 3-thienyl, and 2-, 3- or 4-pyridyl optionally substituted with one or two, substituents which may be the same or different, and are selected from halogen, straight chain alkyl of 1 to 6 carbon atoms, and nitro;
R2 is hydrogen; aryl of 6 to 12 carbon atoms optionally substituted with one or more substituents selected from straight chain alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms, xe2x80x94NO2, and halogen; the heterocyclyl moiety is selected from 2- or 3-furyl, 2- or 3-thienyl, and 2-, 3- or 4-pyridyl; phenylalkyl wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the phenyl moiety is optionally substituted with one or more substituents which may be the same or different and are selected from halogen, straight chain alkyl of 1 to 6 carbon atoms, and nitro; heterocyclylalkyl, wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the heterocyclyl moiety is selected from 2- or 3-furyl, 2- or 3-thienyl, and 2-, 3- or 4-pyridyl;
the optional double bond xe2x80x94 xe2x80x94 xe2x80x94 is a single bond;
where m, u, v, G, Y, R1a, R4, R5a, and R5b are hereinbefore defined;
c)
n is an integer of 2 to 4;
the moiety 
is located at the a or b position of the bicyclic nucleus;
R1 is H;
R2 is H;
R5 is H;
the optional double bond xe2x80x94 xe2x80x94 xe2x80x94 is a single bond;
where m, u, v, G, Y, Axe2x80x94B, R1a, R4, R5a, and R5b are hereinbefore defined;
Among the more preferred groups of compounds of Formula (I) of this invention including pharmaceutically acceptable salts thereof are those in the subgroups
wherein:
a)
n is an integer of 2 to 4;
m is an integer of 1;
v is an integer of 0;
the moiety 
is located at the a or b position of the bicyclic nucleus;
Y is xe2x80x94Oxe2x80x94;
R1 is H;
R2 is H;
R5 is H;
the optional double bond xe2x80x94 xe2x80x94 xe2x80x94 is a single bond;
where u, G, Axe2x80x94B, R1a, R4, R5a, and R5b are hereinbefore defined;
b)
n is an integer of 2 to 4;
the moiety 
is located at the a or b position of the bicyclic nucleus;
R1 is H;
R2 is H;
R5 is H;
G is a moiety selected from the group consisting of: 
where xe2x80x94 xe2x80x94 xe2x80x94, u, v, m, D, Y, R1a, R4, Axe2x80x94B, R5a, and R5b are hereinbefore defined;
c)
n is an integer of 2 to 4;
the moiety 
is located at the a or b-position of the bicyclic nucleus;
R1 is H;
R2 is H;
R5 is H;
Y is xe2x80x94Oxe2x80x94;
G is a moiety selected from the group consisting of: 
where xe2x80x94 xe2x80x94 xe2x80x94, u, V, m, D, R1a, R4, Axe2x80x94B, R5a, and R5b are hereinbefore defined;
d)
n is an integer of 2 to 4;
the moiety 
is located at the b-position of the bicyclic nucleus;
R1 is H;
R2 is H;
R5 is H;
G is a moiety selected from the group consisting of: 
where xe2x80x94 xe2x80x94 xe2x80x94, u, v, m, D, Y, R1a, R4, Axe2x80x94B, R5a, and R5b are hereinbefore defined;
e)
n is an integer of 2 to 4;
the moiety 
is located at the b-position of the bicyclic nucleus;
G is a moiety selected from the group consisting of: 
where xe2x80x94 xe2x80x94 xe2x80x94, u, v, m, Y, R1, R1a, R2, R4, R5, Axe2x80x94B, R5a, and
R5b are hereinbefore defined;
f)
n is an integer of 2 to 4;
R1 is H;
R2 is H;
R5 is H;
Axe2x80x94B is the diradical xe2x80x94CH2xe2x80x94(CH2)mxe2x80x94;
the moiety 
is located at the a or b-position of the bicyclic nucleus;
G is a moiety selected from the group consisting of: 
the optional double bond xe2x80x94 xe2x80x94 xe2x80x94 is a single bond;
where u, v, m, Y, R1a, R4, R5a, and R5b are hereinbefore defined;
Among the specifically preferred compounds of Formula (I) of this invention including pharmaceutically acceptable salts thereof are those set forth below:
4-Methyl-N-({6-[3-(1,4,5,6-tetrahydro-pyrimidin-2-ylamino)-propoxyl]1,2,3,4-tetrahydro-naphthalen-2-yl}-acetyl)-benzenesulfonamide, trifluoroacetic acid salt, and
4-Methyl-N-{[7-(3-guanidino-propoxy)-2-oxo-1,2,3,4-tetrahydro-quinolin-3-yl]-acetyl}-benzenesulfonamide.
In particular, the present invention also provides a method of treatment of diseases characterized by bone resorption of mineralized tissue and by bone loss, resulting from an imbalance between bone resorption and bone formation such as osteoporosis, hypercalcemia of malignancy, osteopenia due to bone metastases, periodontal disease, hyperparathyroidism, periarticular erosions in rheumatoid arthritis, Paget""s disease, immobilization-induced osteopenia and the result of glucocorticoid treatment in warm-blooded animals in need thereof, which comprises administering to said warm-blooded animals, preferably mammals, most preferably humans, an effective amount of a compound of Formulae (I) or (II) or a pharmaceutically acceptable salt thereof.
In addition the present invention also provides a method of blocking or inhibiting bone resorption by antagonizing the xcex1vxcex23 integrin receptor mediated binding of an osteoclast to a bone matrix which comprises administering to warm-blooded animals, preferably mammals, most preferably humans, an effective amount of a compound of general Formulae (I) or (II) or a pharmaceutically acceptable salt thereof. 
wherein:
xe2x80x94 xe2x80x94 xe2x80x94 represents the presence of an optional double bond;
n is an integer of 2 to 5;
v is an integer of 0 or 1;
Axe2x80x94B is a diradical of the formulae: 
m is an integer of 1 or 2;
D is a moiety selected from the group consisting of: 
Y is selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94, 
R1 is hydrogen or straight chain alkyl of 1 to 6 carbon atoms; phenylalkyl wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the phenyl moiety is optionally substituted with one or more substituents which may be the same or different and are selected from hydroxy, amino, halogen, straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, cyano, nitro, alkylamino of 1 to 6 carbon atoms, and dialkylamino of 1 to 6 carbon atoms; heterocyclylalkyl, wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the heterocyclyl moiety is selected from a 5- or 6-membered heterocyclic ring which contains 1 to 3 heteroatoms which may be the same or different, selected from nitrogen, oxygen and sulfur optionally substituted with one or more substituents which may be the same or different, and are selected from hydroxy, amino, halogen, straight chain alkyl of 1 to 6 carbon atoms, cyano and nitro;
R1a is hydrogen or straight chain alkyl of 1 to 6 carbon atoms; phenylalkyl wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the phenyl moiety is optionally substituted with one or more substituents which may be the same or different and are selected from hydroxy, amino, halogen, straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, cyano, nitro, alkylamino of 1 to 6 carbon atoms, and dialkylamino of 1 to 6 carbon atoms;
R2 is hydrogen, xe2x80x94NHR1, or xe2x80x94OR1; aryl of 6 to 12 carbon atoms optionally substituted with one or more substituents selected from straight chain alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms, xe2x80x94S-alkyl of 1 to 6 carbon atoms, cyano, nitro, halogen and phenyl; the heterocyclyl moiety is selected from a 5- or 6-membered heterocyclic ring which contains 1 to 3 heteroatoms which may be the same or different,selected from nitrogen, oxygen and sulfur optionally substituted with one or more substituents which may be the same or different, and are selected from hydroxy, amino, halogen, straight chain alkyl of 1 to 6 carbon atoms, cyano and nitro; phenylalkyl wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the phenyl moiety is optionally substituted with one or more substituents which may be the same or different and are selected from hydroxy, amino, halogen, straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, cyano, nitro, alkylamino of 1 to 6 carbon atoms, and dialkylamino of 1 to 6 carbon atoms; heterocyclylalkyl, wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the heterocyclyl moiety is selected from a 5- or 6-membered heterocyclic ring which contains 1 to 3 heteroatoms which may be the same or different, selected from nitrogen, oxygen and sulfur optionally substituted with one or more substituents which may be the same or different, and are selected from hydroxy, amino, halogen, straight chain alkyl of 1 to 6 carbon atoms, cyano and nitro;
R3 is H, straight chain alkyl of 1 to 6 carbon atoms optionally substituted with a group selected from amino, hydroxyl and carboxyl or branched chain alkyl of 3 to 7 carbon atoms optionally substituted with a group selected from amino, hydroxyl and carboxyl;
G is a moiety selected from the group consisting of: 
u is an integer of 0 or 1;
R4 is straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, alkoxy, or phenylalkyloxy wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the phenyl moiety is optionally substituted with one or more substituents which may be the same or different and are selected from hydroxy, amino, halogen, straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, cyano, nitro, alkylamino of 1 to 6 carbon atoms, and dialkylamino of 1 to 6 carbon atoms;
R5 is hydrogen, straight chain alkyl of 1 to 6 carbon atoms, or phenylalkyl wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the phenyl moiety is optionally substituted with one or more substituents which may be the same or different and are selected from hydroxy, amino, halogen, straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, cyano, nitro, alkylamino of 1 to 6 carbon atoms, and dialkylamino of 1 to 6 carbon atoms;
R5a is hydrogen, straight chain alkyl of 1 to 6 carbon atoms, or phenylalkyl wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the phenyl moiety is optionally substituted with one or more substituents which may be the same or different and are selected from hydroxy, amino, halogen, straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, cyano, nitro, alkylamino of 1 to 6 carbon atoms, and dialkylamino of 1 to 6 carbon atoms;
R5b is hydrogen, straight chain alkyl of 1 to 6 carbon atoms, or phenylalkyl wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the phenyl moiety is optionally substituted with one or more substituents which may be the same or different and are selected from hydroxy, amino, halogen, straight chain alkyl of 1 to 6 carbon atoms, branched chain alkyl of 3 to 7 carbon atoms, cyano, nitro, alkylamino of 1 to 6 carbon atoms, and dialkylamino of 1 to 6 carbon atoms;
with the proviso that Y is not O; n is not 3 or 4; R1, R2, R3 and R5 are not H; D is not xe2x80x94OR3; G is not 
xe2x80x94 xe2x80x94 xe2x80x94 is not a single bond;
a)when v is 0 and substitution is at position a;
with the additional proviso that n is not 2,3 or 4; G is not 
xe2x80x94 xe2x80x94 xe2x80x94 is not a single bond; v is not 1; Axe2x80x94B is not 
D is not xe2x80x94OR3;
a) when Y is xe2x80x94Oxe2x80x94; R1, R2, R3 and R5 are H; and substitution is at position a;
with the still further proviso that when Axe2x80x94B is the moiety 
the moiety 
is located at the a,b or c positions of the bicyclic nucleus;
and with the additional proviso that the optional double bond xe2x80x94 xe2x80x94 xe2x80x94 is a single bond when Axe2x80x94B is the diradical xe2x80x94CH2xe2x80x94(CH2)mxe2x80x94;
or a pharmaceutically acceptable salt thereof.
For the compounds defined for Formulae (I) or (II) above and referred to herein, unless otherwise noted, the following terms are defined:
The term halogen may be selected from fluorine, chlorine, bromine and iodine, unless otherwise specified.
Phenyl as used herein refers to a 6-membered aromatic ring.
The term alkoxy means an alkyl group having a straight chain alkyl group attached through an oxygen bridge and including for example methoxy, ethoxy, n-propoxy, n-butoxy, and the like.
The term aryl when used alone means a homocyclic aromatic radical, whether or not fused, having 6 to 10 carbon atoms. Preferred aryl groups include phenyl, alpha-naphthyl and beta-naphthyl and the like optionally substituted.
The term heterocyclyl means an optionally substituted monocyclic heteroaromatic ring. Preferred are 2- or 3-furyl, 2- or 3-thienyl, 2-, 3- or 4-pyridyl.
The range of carbon atoms defines the total number of carbon atoms in the substituent group.
The compounds of Formulae (I) or (II) of the present invention can be used in the form of salts derived from pharmaceutically or physiologically acceptable acids or bases. These salts include, but are not limited to, the following: salts with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and, as the case may be, such organic acids as acetic acid, oxalic acid, succinic acid, and maleic acid. Other salts include salts with alkali metals or alkaline earth metals, such as sodium, potassium, calcium or magnesium or with organic bases. The compounds can also be used in the form of esters, carbamates and other conventional xe2x80x9cpro-drugxe2x80x9d forms, which, when administered in such form, convert to the active moiety in vivo.
Among the preferred groups of compounds of Formula (II) of this invention including pharmaceutically acceptable salts thereof are those in the subgroups
wherein:
a)
D is the moiety 
R3 is H;
where xe2x80x94 xe2x80x94 xe2x80x94, n, m, u, v, G, Y, R1, R1a, R2, R4, R5a, and R5b are hereinbefore defined;
b)
n is an integer of 2 to 4;
v is an integer of 0;
the moiety 
is located at the a or b position of the bicyclic nucleus;
R1 is hydrogen or straight chain alkyl of 1 to 6 carbon atoms; phenylalkyl wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the phenyl moiety is optionally substituted with one or two substituents which may be the same or different and are selected from halogen, straight chain alkyl of 1 to 6 carbon atoms, and nitro; heterocyclylalkyl, wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the heterocyclyl moiety is selected from 2- or 3-furyl, 2- or 3-thienyl, and 2-, 3- or 4-pyridyl optionally substituted with one or two, substituents which may be the same or different, and are selected from halogen, straight chain alkyl of 1 to 6 carbon atoms, and nitro;
R2 is hydrogen; aryl of 6 to 12 carbon atoms optionally substituted with one or more substituents selected from straight chain alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms, nitro, and halogen; the heterocyclyl moiety is selected from 2- or 3-furyl, 2- or 3-thienyl, and 2-, 3- or 4-pyridyl; phenylalkyl wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the phenyl moiety is optionally substituted with one or more substituents which may be the same or different and are selected from halogen, straight chain alkyl of 1 to 6 carbon atoms, and nitro; heterocyclylalkyl, wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the heterocyclyl moiety is selected from 2- or 3-furyl, 2- or 3-thienyl, and 2-, 3- or 4-pyridyl;
the optional double bond xe2x80x94 xe2x80x94 xe2x80x94 is a single bond;
where m, u, G, Y, D, Axe2x80x94B, R1a, R3, R4, R5a, and R5b are hereinbefore defined;
c)
n is an integer of 2 to 4;
v is an integer of 0;
the moiety 
is located at the a or b position of the bicyclic nucleus;
Axe2x80x94B is the diradical xe2x80x94CH2xe2x80x94(CH2)mxe2x80x94;
R1 is hydrogen or straight chain alkyl of 1 to 6 carbon atoms; phenylalkyl wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the phenyl moiety is optionally substituted with one or two substituents which may be the same or different and are selected from halogen, straight chain alkyl of 1 to 6 carbon atoms, and nitro; heterocyclylalkyl, wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the heterocyclyl moiety is selected from 2- or 3-furyl, 2- or 3-thienyl, and 2-, 3- or 4-pyridyl optionally substituted with one or two, substituents which may be the same or different, and are selected from halogen, straight chain alkyl of 1 to 6 carbon atoms, and nitro;
R2 is hydrogen; aryl of 6 to 12 carbon atoms optionally substituted with one or more substituents selected from straight chain alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms, xe2x80x94NO2, and halogen; the heterocyclyl moiety is selected from 2- or 3-furyl, 2- or 3-thienyl, and 2-, 3- or 4-pyridyl; phenylalkyl wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the phenyl moiety is optionally substituted with one or more substituents which may be the same or different and are selected from halogen, straight chain alkyl of 1 to 6 carbon atoms, and nitro; heterocyclylalkyl, wherein the alkyl moiety is a straight chain alkyl of 1 to 6 carbon atoms and the heterocyclyl moiety is selected from 2- or 3-furyl, 2- or 3-thienyl, and 2-, 3- or 4-pyridyl;
the optional double bond xe2x80x94 xe2x80x94 xe2x80x94 is a single bond;
where m, u, G, Y, D, R1a, R3, R4, R5a, and R5b are hereinbefore defined;
d)
n is an integer of 2 to 4;
v is an integer of 0;
the moiety 
is located at the a or b position of the bicyclic nucleus;
R1 is H;
R2 is H;
R5 is H;
the optional double bond xe2x80x94 xe2x80x94 xe2x80x94 is a single bond;
where m, u, G, Y, Axe2x80x94B, D, R1a, R3, R4, R5a, and R5b are hereinbefore defined;
e)
n is an integer of 2 to 4;
v is an integer of 0;
the moiety 
is located at the a or b position of the bicyclic nucleus;
R1 is H;
R2 is H;
Axe2x80x94B is the diradical xe2x80x94CH2xe2x80x94(CH2)mxe2x80x94;
Y is xe2x80x94Oxe2x80x94;
the optional double bond xe2x80x94 xe2x80x94 xe2x80x94 is a single bond;
where m, u, D, G, R1a, R3, R4, R5a, and R5b are hereinbefore defined;
f)
n is an integer of 2 to 4;
v is an integer of 0;
the moiety 
is located at the a or b position of the bicyclic nucleus;
R1 is H;
R2 is H;
R5 is H;
Y is xe2x80x94Oxe2x80x94;
where xe2x80x94 xe2x80x94 xe2x80x94, u, G, D, Axe2x80x94B, R1a, R3, R4, R5a, and R5b are hereinbefore defined;
Among the more preferred groups of compounds of Formula (II) of this invention including pharmaceutically acceptable salts thereof are those in the subgroups wherein:
a)
n is an integer of 2 to 4;
m is an integer of 1;
v is an integer of 0;
the moiety 
is located at the a or b position of the bicyclic nucleus;
Y is xe2x80x94Oxe2x80x94;
R1 is H;
R2 is H;
R5 is H;
the optional double bond xe2x80x94 xe2x80x94 xe2x80x94 is a single bond;
where u, G, D, Axe2x80x94B, R1a, R3, R4, R5a, and R5b are hereinbefore defined;
b)
n is an integer of 2 to 4;
m is an integer of 2;
v is an integer of 0;
the moiety 
is located at the a or b position of the bicyclic nucleus;
Y is xe2x80x94Oxe2x80x94;
R1 is H;
R2 is H;
R5 is H;
the optional double bond xe2x80x94 xe2x80x94 xe2x80x94 is a single bond;
where u, G, D, Axe2x80x94B, R1a, R3, R4, R5a, and R5b are hereinbefore defined;
Among the particularly preferred groups of compounds of Formula (II) of this invention including pharmaceutically acceptable salts thereof are those in the subgroups wherein:
a)
n is an integer of 2 to 4;
v is an integer of 0;
the moiety 
is located at the a or b position of the bicyclic nucleus;
R1 is H;
R2 is H;
R5 is H;
G is a moiety selected from the group consisting of: 
where xe2x80x94 xe2x80x94 xe2x80x94, u, m, D, Y, R1a, R3, R4, Axe2x80x94B, R5a, and R5b are hereinbefore defined;
b)
n is an integer of 2 to 4;
v is an integer of 0;
the moiety 
is located at the a or b-position of the bicyclic nucleus;
R1 is H;
R2 is H;
R5 is H;
Y is xe2x80x94Oxe2x80x94;
G is a moiety selected from the group consisting of: 
where xe2x80x94 xe2x80x94 xe2x80x94, u, m, D, R1a, R3, R4, Axe2x80x94B, R5a, and R5b are hereinbefore defined;
c)
n is an integer of 2 to 4;
v is an integer of 0;
the moiety 
is located at the b-position of the bicyclic nucleus;
R1 is H;
R2 is H;
R5 is H;
G is a moiety selected from the group consisting of: 
where xe2x80x94 xe2x80x94 xe2x80x94, u, m, D, Y, R1a, R3, R4, Axe2x80x94B, R5a, and R5b are hereinbefore defined;
d)
n is an integer of 2 to 4;
the moiety 
is located at the a or b-position of the bicyclic nucleus;
G is a moiety selected from the group consisting of: 
D is moiety 
where xe2x80x94 xe2x80x94 xe2x80x94, u, v, m, Y, R1, R1a, R2, R4, R5, Axe2x80x94B, R5a, and R5b are hereinbefore defined;
e)
n is an integer of 2 to 4;
the moiety 
is located at the b-position of the bicyclic nucleus;
G is a moiety selected from the group consisting of: 
D is a moiety 
where xe2x80x94 xe2x80x94 xe2x80x94, u, v, m, Y, R1, R1a, R2, R4, R5, Axe2x80x94B, R5a, and R5b are hereinbefore defined;
f)
n is an integer of 2 to 4;
R1 is H;
R2 is H;
R5 is H;
Axe2x80x94B is the diradical xe2x80x94CH2xe2x80x94(CH2)mxe2x80x94;
the moiety 
is located at the a or b-position of the bicyclic nucleus;
G is a moiety selected from the group consisting of: 
D is a moiety 
the optional double bond xe2x80x94 xe2x80x94 xe2x80x94 is a single bond;
where u, v, m, Y, R1a, R4, R5a, and R5b are hereinbefore defined;
g)
n is an integer of 2 to 4;
the moiety 
is located at the a or b-position of the bicyclic nucleus;
G is a moiety selected from the group consisting of: 
D is a moiety xe2x80x94OR3;
R3 is H;
where xe2x80x94 xe2x80x94 xe2x80x94, u, v, m, Y, R1, R1a, R2, R4, R5, Axe2x80x94B, R5a, and R5b are hereinbefore defined;
Among the specifically preferred compounds of Formula (II) of this invention including pharmaceutically acceptable salts thereof are those set forth below:
[6-(3-Guanidinopropoxy)-1,2,3,4-tetrahydro-napthalen-2-yl]-acetic acid ethyl ester,
[6-(3-Guanidino-propoxy)-1,2,3,4-tetrahydro-napthalen-2-yl]-acetic acid trifluoroacetate,
[7-(3-Guanidino-propoxy)-1,2,3,4-tetrahydro-napthalen-2-yl]-acetic acid trifluoroacetate,
[2-(2-Guanidino-ethoxy)-6,7,8,9-tetrahydro-5H-benzocyclohepten-6-yl]-acetic acid hydrochloride,
[2-(3-Guanidino-propoxy)-6,7,8,9-tetrahydro-5H-benzocyclohepten-6-yl]-acetic acid trifluoroacetate,
[2-(4-Guanidino-butoxy)-6,7,8,9-tetrahydro-5H-benzocyclohepten-6-yl]-acetic acid trifluoroacetate,
[7-(4-Guanidino-butoxy)-2-oxo-1,2,3,4-tetrahydro-quinolin-3-yl]-acetic acid trifluoroacetate,
[6-(4-Guanidino-butoxy)-2-oxo-1,2,3,4-tetrahydro-quinolin-3-yl]-acetic acid trifluoroacetate,
[7-(2-Guanidino-ethoxy)-2-oxo-1,2,3,4-tetrahydro-quinolin-3-yl]-acetic acid Hydrochloride,
[7-(3-Guanidino-propoxy)-2-oxo-1,2,3,4-tetrahydro-quinolin-3-yl]-acetic acid Hydrochloride,
[7-(4-Guanidino-but-1-ynyl)-2-oxo-1,2,3,4-tetrahydro-quinolin-3-yl]-acetic acid Hydrochloride,
[7-(5-Guanidino-pent-1-ynyl)-2-oxo-1,2,3,4-tetrahydro-quinolin-3-yl]-acetic acid Trifluoroacetate,
[7-(4-Guanidino-but-1-enyl)-2-oxo-1,2,3,4-tetrahydro-quinolin-3-yl]-acetic acid Trifluoroacetate,
{6-[3-(Pyrimidin-2-ylamino)-propoxy]-1,2,3,4-tetrahydro-naphthalen-2-yl}-acetic acid,
{6-[3-(1,4,5,6-Tetrahydro-pyrimidin-2-ylamino)-propoxy]-1,2,3,4-tetrahydro-naphthalen-2-yl}-acetic acid,
{6-[3-(1,4,5,6-Tetrahydro-pyrimidin-2-ylamino)-propoxy]-1,2,3,4-tetrahydro-naphthalen-2-yl}-acetic acid methyl ester bis(hydrochloride),
{6-[3-(1,4,5,6-Tetrahydro-pyrimidin-2-ylamino)-propoxy]-1,2,3,4-tetrahydro-naphthalen-2-yl}-acetic acid ethyl ester, acetic acid salt,
4-Methyl-N-({6-[3-(1,4,5,6-tetrahydro-pyrimidin-2-ylamino)-propoxyl]1,2,3,4-tetrahydro-naphthalen-2-yl]-acetyl)-benzenesulfonamide, trifluoroacetic acid salt,
[6-(3-Guanidino-propoxy)-2-oxo-1,2,3,4-tetrahydro-quinolin-3-yl]-acetic acid,
3-[7-(2-Guanidino-ethoxy)-2-oxo-1,2,3,4-tetrahydro-quinolin-3-yl]-propionic acid,
3-[7-(3-Guanidino-propoxy)-2-oxo-1,2,3,4-tetrahydro-quinolin-3-yl]-propionic acid,
[8-(3-Guanidino-propoxy)-2-oxo-1,2,3,4-tetrahydro-quinolin-3-yl]-acetic acid,
[8-(4-Guanidino-butoxy)-2-oxo-1,2,3,4-tetrahydro-quinolin-3-yl]-acetic acid,
[1-Benzyl-7-(3-guanidino-propoxy)-2-oxo-1,2-dihydro-quinolin-3-yl]-acetic acid Trifluoroacetate,
[7-(5-Guanidino-pent-1-enyl)-2-oxo-1,2,3,4-tetrahydro-quinolin-3-yl]-acetic acid Trifluoroacetate,
[7-(4-Guanidino-butyl)-2-oxo-1,2,3,4-tetrahydro-quinolin-3-yl]-acetic acid Trifluoroacetate,
[7-(5-Guanidino-pentyl)-2-oxo-1,2,3,4-tetrahydro-quinolin-3-yl]-acetic acid Trifluoroacetate,
[1-Ethyl-7-(3-guanidino-propoxy)-2-oxo-1,2-dihydro-quinolin-3-yl]-acetic acid Trifluoroacetate
[1-Benzyl-7-(3-guanidino-propoxy)-2-oxo-1,2-dihydro-quinolin-3-yl]-acetic acid
[1-Ethyl-7-(3-guanidino-propoxy)-2-oxo-1,2,3,,4-tetrahydro-quinolin-3-yl]-acetic acid Trifluoroacetate,
[1-Benzyl-7-(3-guanidino-propoxy)-2-oxo-1,2,3,4-tetrahydro-quinolin-3-yl]-acetic acid,
[7-(4-Guanidino-butoxy)-2-oxo-1,2-dihydro-quinolin-3-yl]-acetic acid Hydrochloride,
[7-(2-Guanidino-ethoxy)-2-oxo-1,2-dihydro-quinolin-3-yl]-acetic acid,
[7-(3-Guanidino-propoxy)-2-oxo-1,2-dihydro-quinolin-3-yl]-acetic acid Trifluoroacetate,
[7-(2-Guanidino-ethylcarbamoyl)-2-oxo-1,2,3,4-tetrahydro-quinolin-3-yl]-acetic acid Hydrochloride,
[7-(3-Guanidino-propylcarbamoyl)-2-oxo-1,2,3,4-tetrahydro-quinolin-3-yl]-acetic acid Hydrochloride,
{6-[3-(Pyrimidin-2-ylamino)-propoxy]-1,2,3,4-tetrahydro-naphthalen-2-yl}-acetic acid methyl ester,
3-[7-(2-Guanidino-ethoxy)-2-oxo-1,2-dihydro-quinolin-3-yl]propionic acid nitric acid salt,
4-Methyl-N-{[7-(3-guanidino-propoxy)-2-oxo-1,2,3,4-tetrahydro-quinolin-3-yl]-acetyl}-benzenesulfonamide and
[8-(5-Guanidino-pentoxy)-2-oxo-1,2,3,4-tetrahydro-quinolin-3-yl]-acetic acid.
Some of the compounds of the hereinafter described schemes have centers of asymmetry. The compounds may, therefore, exist in at least two and often more stereoisomeric forms. The present invention encompasses all stereoisomers of the compounds whether free from other stereoisomers or admixed with other stereoisomers in any proportion and thus includes, for instance, racemic mixture of enantiomers as well as the diastereomeric mixture of isomers. The absolute configuration of any compound may be determined by conventional X-ray crystallography.
The present invention accordingly provides a pharmaceutical composition which comprises a compound of Formulae (I) or (II) of this invention in combination or association with a pharmaceutically acceptable carrier. In particular, the present invention provides a pharmaceutical composition which comprises an effective amount of a compound of this invention and a pharmaceutically acceptable carrier.
The compounds of the present invention may be prepared according to the following reaction schemes.
In Scheme I, bicyclic ketone 1 where Y is xe2x80x94Oxe2x80x94, Axe2x80x94B is the diradical xe2x80x94CH2xe2x80x94(CH2)mxe2x80x94, and m is 1 or 2 is reacted with tri(C1-C6)alkyl phosphonoacetate 2 where v and R2 are hereinbefore defined in the presence of potassium tert-butoxide to give olefin 3. Tri(C1-C6)alkyl phosphonoacetate 2 may be prepared using the conditions as described in U.S. Pat. Nos. 5,312,828 and 5,473,092. Bicyclic ketone 1 where m is 1 can be prepared from dimethoxynaphthalene as described by S. Copinga et al., J. Med. Chem., 36, 2891-2898 (1993) or as described by A. Cordi et al, J.Med.Chem., 38, 4056-4069(1995) and where m is 1 or 2 as described in G. Pandey et al., Tetrahedron Lett. 1993, 34, 6631-6634. Catalytic hydrogenation of olefin 3 in the presence of palladium-on-carbon affords ester 4. Treating ester 4 with boron tribromide in methylene chloride at 0xc2x0 C. gives phenol 5 where Y is xe2x80x94Oxe2x80x94, Axe2x80x94B is the diradical xe2x80x94CH2xe2x80x94(CH2)m, and v, m and R2 are hereinbefore defined. Alkylation of ester 4 where v is 0 or 1 with R2X where R2 is hereinbefore defined provided R2 is not H, in the presence of a base such as sodium methoxide and where X is a leaving group which includes but is not limited to xe2x80x94Cl, xe2x80x94Br, xe2x80x94I and methanesulfonyl gives ester 6. Treating ester 6 with boron tribromide in methylene chloride at 0xc2x0 C. gives phenol 7 where Y is xe2x80x94Oxe2x80x94, R2 is hereinbefore defined excluding hydrogen, Axe2x80x94B is the diradical xe2x80x94CH2xe2x80x94(CH2)mxe2x80x94, and v and m are hereinbefore defined. 
As described in Scheme II, nitrobenzaldehyde 8 where R is straight chain alkyl of 1 to 6 carbon atoms is reacted with diester 9, in acetic acid where v and R2 are hereinbefore defined and W is a moiety 
to give the corresponding diester 10 where R, R2 and v are hereinbefore defined. Diester 9 where v is an integer of 0, R2 is H and W is 
can be prepared in situ from a distraight chain lower alkyl of 1 to 6 carbon atoms maleate and triphenyl phosphine in acetic acid, according to the modified method of Kadin, S. B. and Lamphere, C. H., J. Org. Chem., 49, 4999 (1984), and in the case where v is an integer of 1 from ethyl xcex1-bromo-glutanate (E. Schwenk and D. Papa, J. Am. Chem. Soc., 70 3626-3627 (1948)). Diester 9 where v and R2 are hereinbefore defined and W is 
may be prepared using the conditions as described in P. G. Baraldi et al, J.Chem. Soc., Perkin Trans. I, 2501-2505(1984) and GB1423495. Reduction of the nitro and olefinic groups of diester 10 by catalytic hydrogenation (10% Pd/C) followed by spontaneous cyclization gives tetrahydroquinolinone 11 where R, R2 and v are hereinbefore defined and the moiety Axe2x80x94B is the diradical 
where R5 is H. Reduction of the nitro group of diester 10 using zinc in 12N HCl-ethyl alcohol followed by spontaneous cyclization gives substituted (1,2-dihydro-3-yl)alkanoate ester 12 where R, R2 and v are hereinbefore defined and R5 is H. Alternatively, as also shown in Scheme II, substituted (1,2-dihydro-3-yl)alkanoate ester 12 where R is hereinbefore defined may be converted to phenol 13 by reaction with borontribromide followed by catalytic reduction in the presence of palladium-on-carbon to give phenol 14 where R2 and v are hereinbefore defined and the moiety Axe2x80x94B is the diradical 
where R5 is H. Catalytic reduction of substituted (1,2-dihydro-3-yl) alkanoate ester 12 where R, R2 and v are hereinbefore defined and R5 is H in the presence of palladium-on-carbon affords substituted tetrahydroquinolinone 11 where R, R2 and v are hereinbefore defined and R5 is H.
Again, referring to Scheme II, (1,2-dihydro-3-yl)alkanoate ester 12 where R5 is H is alkylated with R5 X where R5 is hereinbefore defined excluding hydrogen and X is a leaving group which includes but is not limited to xe2x80x94Cl, xe2x80x94Br, xe2x80x94I and methanesulfonyl in the presence of potassium bis(trimethylsilyl)amide (KN(TMS)2) to give ester 16. Treating ester 16 with boron tribromide can afford phenol 13. 
Again referring to Scheme II, tetrahydroquinolinone 11 where R and R2 are hereinbefore defined and R5 is H is alkylated with R5X where R5 is hereinbefore defined excluding hydrogen and X is a leaving group which includes but is not limited to xe2x80x94Cl, xe2x80x94Br, xe2x80x94I and methanesulfonyl in the presence of potassium bis(trimethylsilyl)amide (KN(TMS)2) to give ester 15. Treating ester 15 with boron tribromide can afford phenol 17.
Additionally, a method of preparing substituted (1,2-dihydro-3-yl)alkanoate ester 12 is shown in Scheme III using the method as described by O. Meth-Cohn et al, J. Chem. Soc. Perkin I, 1537-1543 (1981). Methoxy substituted aniline 18 is reacted with acid chloride 19 where v and R2 are hereinbefore defined to give amide 20. Acid chloride 19 is prepared from the corresponding half acid-ester by reaction with thionyl chloride or oxalyl chloride. Further reaction of amide 20 where v and R2 are hereinbefore defined with phosphorous oxychloride in N,N-dimethylformamide affords 2-chloro-substituted quinoline 21. Hydrolysis of 2-chloro-substituted quinoline 21 with aqueous HCl in methanol affords substituted (1,2-dihydro-3-yl)alkanoate ester 12 (R is CH3), where v and R2 are hereinbefore defined. 
As described in Scheme IV, aldehyde 22 is reacted with tri(C1-C6)alkyl phosphonoacetate 2 where v is 0 and R2 is H in the presence of sodium hydride in tetrahydrofuran to give ester 23 which is hydrolyzed with 12N HCl to afford (1,2-dihydro-3-yl)alkanoate ester 24. reduction of (1,2-dihydro-3-yl)alkanoate ester 24 with hydrogen in the presence of 10% Pd/C in acetic acid affords tetrahydroquinolinone 25 which is further reacted with BBr3 in methylene chloride to give phenol 14a where V is 1, R2is H and R5 is H. 
As shown in Scheme V, substituted amino alcohol 26 where R1 and n are hereinbefore defined is converted to tert-butyl carbamate 27 by reaction with di-tert-butyl dicarbonate in the presence of potassium carbonate and which is further reacted with carbon tetrabromide in the presence of triphenylphosphine to give (bromoalkyl)carbamic acid tert-butyl ester 28 where R1 and n are hereinbefore defined. 
As shown in Scheme VI, independent alkylation of phenol 5, 7, 13, or 14, where Y is xe2x80x94Qxe2x80x94, Axe2x80x94B, m, V, are hereinbefore defined and R2 and R5 are as defined for each phenol and xe2x80x94 xe2x80x94 xe2x80x94 is an optional double bond when Axe2x80x94B is 
with (bromoalkyl) carbamic acid tert-butyl ester 28 where R1 and n are hereinbefore defined using sodium ethoxide in N,N-dimethylformamide gives ether 29 where R1, R2, R5, n, v, Axe2x80x94B and m are hereinbefore defined and Y is xe2x80x94Oxe2x80x94. Removal of the tert-butyl ester of ether 29 with trifluoroacetic acid (TFA) gives amine 30. 
An alternative to using bromoalkylcarbamic acid t-butylester 28 is shown in Scheme VII where independent alkylation of phenol 5, 7, 13,or 14 with N-(brormoalkyl)-phthalimide 31 where n is hereinbefore defined, in the presence of sodium hydride in N,N-dimethylformamide affords ester 32 where Y is xe2x80x94Oxe2x80x94 and v, n, m, Axe2x80x94B, R2 and R5 are hereinbefore defined and xe2x80x94 xe2x80x94 xe2x80x94 is an optional double bond when Axe2x80x94B is 
The phthalimide blocking group of ester 32 is removed by reaction with hydrazine in isopropyl alcohol to give amine 33 where Y is xe2x80x94Oxe2x80x94, and R5, R2, v, n, m and Axe2x80x94B are hereinbefore defined. Ester 33 may be alkylated with R1X where R1 is not H in the presence of base to give amine 30. 
As outlined in Scheme VIII, phenol 5, 7, 13, or 14, where Y is xe2x80x94Oxe2x80x94 and Axe2x80x94B, m, and v are hereinbefore defined and R2 and R5 are as defined for each phenol which can be independently reacted with trifluoro-methane sulfonic anhydride (Tf2O) to give triflate 34. Palladium mediated coupling of triflate 34 with tert-butyloxycarbonyl (Boc) protected acetylene 35 where n is hereinbefore defined and Y is:
xe2x80x94Cxe2x89xa1Cxe2x80x94
gives acetylene 36 where Y is:
xe2x80x94Cxe2x89xa1Cxe2x80x94,
and Axe2x80x94B, R2, R5, n, m and v are hereinbefore defined. Reduction of acetylene 36 with hydrogen in the presence of Lindlar catalyst gives olefin 37 where Y is xe2x80x94CHxe2x95x90CHxe2x80x94 and Axe2x80x94B, R5 R21, v, n, m are hereinbefore defined and xe2x80x94 xe2x80x94 xe2x80x94 is a single bond. Olefin 37 can be reacted with trifluoroacetic acid to give amine 38 where Y is xe2x80x94CHxe2x95x90CHxe2x80x94 and Axe2x80x94B, R2, R5, n, m and v are hereinbefore defined and xe2x80x94 xe2x80x94 xe2x80x94 is a single bond. Acetylene 36 can be reacted with trifluoroacetic acid to give amine 39 where Y is
xe2x80x94Cxe2x89xa1Cxe2x80x94
and Axe2x80x94B, R2, R5, n, m and v are hereinbefore defined. Reduction of amine 39 in the presence of palladium-on-carbon and hydrogen in acetic acid gives amine 40 where Y is xe2x80x94CH2xe2x80x94CH2xe2x80x94 and Axe2x80x94B, R2, R5, n, m and v are hereinbefore defined. Independent alkylation of amines 38, 39, and 40 with R1X where R1 is hereinbefore defined, provided that R1 is not H, in the presence of base such as sodium methoxide and X is a leaving group gives amines 41, 42, and 43 respectively.
Compounds of Formulae (I) or (II) wherein Y is 
where R1a is hereinbefore defined;
Axe2x80x94B is the diradical 
R5 is H straight chain alkyl of 1 to 6 carbon atoms and substituted benzyl, n is an integer from 2 to 4 and v is an integer of 0 or 1 may be prepared as shown in Scheme IX, where tert-butyl-3-nitro-4-bromomethyl-benzoate 44 (Y. Kashman and J. A. Edwards, J. org. Chem. 43, 1538-1540 (1978)) is first reacted with pyridine in ethanol followed by further reaction with p-nitrosodimethylamine in the presence of aqueous 2.0 N sodium hydroxide followed by further treatment with aqueous 6 N sulfuric acid affords aldehyde 45 using the conditions described in organic Synthesis, Collective Volume V, page 825. Reaction of aldehyde 45 with diester 9 where v and R2 are hereinbefore defined gives tert-butyl ester 46. Catalytic hydrogenation of tert-butyl ester 46 in the presence of 10% Pd/C and spontaneous cyclization gives lactam 47 where Axe2x80x94B is the diradical 
where R and v are hereinbefore defined and R5 is H. Alkylation of lactam 47 with R5X where R5 is hereinbefore defined excluding H and X is a leaving group hereinbefore defined in the presence of base can form ester 48. Hydrolysis of lactam 47 and ester 48 with aqueous 4 N hydrochloric acid in dioxane gives carboxylic acid 49 where R2, v and R5 are hereinbefore defined. Reaction of carboxylic acid 49 with 1-hydroxybenzotriazole hydrate (HOBT) and carbodiimide 50 where n is hereinbefore defined gives ester 51 where Axe2x80x94B is the diradical 
and R1a, R2, R5, n and v are hereinbefore defined. The N-tertbutoxycarbonyl blocking group on ester 51 is removed by stirring with trifluoroacetic acid in methylene chloride to give amine 52. Alkylation of amine 52 with R1X where R1 is hereinbefore defined excluding H can afford amine 53. 
Compounds of Formulae (I) or (II) wherein Y is 
Axe2x80x94B is the diradical xe2x80x94CH2xe2x80x94(CH2)mxe2x80x94, R1a and m are hereinbefore defined may be prepared as shown in Scheme X, where phenol 5 can be reacted with trifluoromethane sulfonic anhydride (Tf2O) to give triflate 54 which can be further reacted with CO in the presence of Pdo followed by treatment with aqueous base to give carboxylic acid 55 where Axe2x80x94B is the diradical xe2x80x94CH2xe2x80x94(CH2)m, and m, v and R2 are hereinbefore defined. Reaction of carboxylic acid 55 with 1-hydroxybenzo-triazole hydrate (HOBT) and carbodiimide 50 where n and R1a are hereinbefore defined can give ester 56. The N-tertbutoxy-carbonyl blocking group on ester 56 may be removed by stirring with trifluoroacetic acid in methylene chloride to form amine 57 where n, v, R1a and R2 are hereinbefore defined, Y is 
and Axe2x80x94B is the diradical xe2x80x94CH2xe2x80x94(CH2)mxe2x80x94. Alkylation of amine 57 with R1X where R1 is hereinbefore defined excluding H can afford amine 58. 
As shown in Scheme XI, amines 30, 38, 39, 40, 41, 42, 43, 52, 53, 57, and 58 are independently reacted with a G-reagent 59 where G is hereinbefore defined using the conditions and methods as described in WO0 97/36862, WO 97/33887, WO 97/37655 and CA2199923 with the exception where G is pyrimidine, the preferred method is to in situ activate amines 30, 38, 39, 40, 41, 42, 43, 52, 53, 57, and 58 with trimethylsilyl chloride in the presence of 2-bromo-pyrimidine in refluxing anhydrous 1,4-dioxane to give ester 60. G-reagent 59 includes but is not limited to those in Table A. In particular, alkylation of amines 30, 38, 39, 40, 41, 42, 43, 52, 53, 57, and 58 with 2-methylthio-3,4,5,6-tetrahydro-pyrimidine hydroiodide, a G-reagent 59, using the conditions as described (WO 96/37492 Example 83) can give ester 60 where G is 
Alternatively, condensation of amines 30, 38, 39, 40, 41, 42, 43, 52, 53, 57, and 58 with N,Nxe2x80x2-bis(tert-butoxycarbonyl)-2-(1H)-tetrahydropyrimidine-thione followed by deprotection with hydrochloric acid can give ester 60 where G is 
Independent base hydrolysis of ester 60 with aqueous base gives carboxylic acid 61. Suitable bases include sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate and potassium carbonate.
Again referring to Scheme XI, carboxylic acid 61 was reacted with substitutedbenzenesulfonamide 62 
where R5a and R5b are hereinbefore defined in the presence of 1-[3-(dimethylamino)propyl]-3-ethyl-carbodiimide hydrochloride, dimethylaminopyridine and N,N-dimethylformamide (DMF) to give substitutedbenzenesulfonamide 63 and v, n, m, G, Axe2x80x94B, R1, R1a, R2, R5, R5a and R5b are hereinbefore defined.
Reduction of carboxylic acid 61 where G is the selected moiety 
and where Y is xe2x80x94CHxe2x95x90CHxe2x80x94, or
xe2x80x94Cxe2x89xa1Cxe2x80x94,
in the presence of hydrochloric acid, acetic acid and an alcohol (C1-C6)OH followed by reaction with an alcohol (C1-C6)OH in the presence of hydrochloric acid gives an ester where G is reduced to the tetrahydropyrimidine moiety 
Y is reduced to xe2x80x94CH2xe2x80x94CH2xe2x80x94 and the optional double bond xe2x80x94xe2x80x94xe2x80x94 xe2x80x94 xe2x80x94 is also reduced to a single bond and v, n, m, Axe2x80x94B, R1, R1a, R2, and R5 are hereinbefore defined. 
The compounds of the present invention can be prepared readily according to hereinbefore described reaction schemes and hereinafter described examples or modifications thereof using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants which are themselves known to those of ordinary skill in this art, but are not mentioned in greater detail.
The most particularly preferred compounds of the invention are any or all of those specifically set forth in these examples. These compounds are not, however, to be construed as forming the only genus that is considered as the invention, and any combination of the compounds or their moieties may itself form a genus. The following examples further illustrate details for the preparation of the compounds of the present invention.
Representative compounds of the present invention were evaluated in the following pharmacological test procedures which measured Vitronectin Receptor (xcex1vxcex23) Binding, Osteopontin (xcex1vxcex23)Cell Attachment, Osteoclast Bone Pitting, PTH-induced hypercalcemia and ADPxe2x80x94Induced Platelet Aggregation and which further show that the compounds of the present invention selectively antagonize the (xcex1vxcex23) integrin while not displaying ADP-induced platelet aggregation mediated by a fibrinogen (xcex1IIbxcex23) integrin.
Vitronectin Receptor (xcex1vxcex23) Binding Test Procedure
Measuring the effect of compounds on the xcex1vxcex23-ligand interaction.
Reagents
Plasma Membrane Isolation: 15 confluent T150 flasks of 512P5 cells (xcex1vxcex23xe2x80x94overexpressing cell line) were washed 2xc3x97 with Dulbecco""s phosphate buffered saline (D-PBS) without calcium or magnesium, pH 7.1. Cells were harvested with 10 mL of trypsin/EDTA and collected by centrifugation. The cell pellet was washed 2xc3x97 with 0.5 mg/mL of soybean trypsin inhibitor, and resuspended at 10% weight/volume in homogenization buffer (25 mM Tris-HCl, pH=7.4; 250 mM sucrose). The cell suspension was homogenized with 2xc3x9730 seconds bursts of a Polytron homogenizer. The homogenate was centrifuged at 3000 g for 10 minutes at 4xc2x0 C. The supernatant was collected, measured, and made 100 mM in NaCl and 0.2 mM in MgSO4. The supernatant was centrifuged at 22,000 g for 20 minutes at 4xc2x0 C., the pellet was resuspended in 7 mL of membrane buffer (25 mM Tris-HCl, pH=7.4; 100 mM NaCl; 2 mM MgCl2) by 5 strokes of a Dounce homogenizer (tight pestle) and recentrifuged at 22,000 g for 20 minutes at 4xc2x0 C. The pellet was resuspended in 0.5 mL/flask of membrane buffer (stock membranes) and frozen at xe2x88x9280xc2x0 C. Prior to use, stock membranes were Dounce homogenized and diluted 2 xcexcL to 1000 xcexcL in membrane buffer.
Compound Dilution: The stock compounds were dissolved in an appropriate vehicle (typically DMSO) and subsequently diluted in assay buffer composed as follows: 25 mM Tris-HCl (pH=7.4), 100 mM NaCl, 2 mM MgCl2, 0.1% BSA.
Plate Preparation
Wells of Multiscreen-FB assay plates (Millipore MAFB NOB 50) were blocked with 150 xcexcL of 0.1% polyethylenimine for 2 hours at 4xc2x0 C. Following incubation the wells were aspirated and washed with isotonic saline solution.
Binding Assay
125 xcexcL of assay buffer was added to each well. Next, 25 xcexcL of labeled ligand was added to each well. 25 xcexcL of unlabeled ligand was added to non-specific binding wells (NSB). 25 xcexcL of assay buffer was added to all other wells. 2 xcexcL of compound was added to appropriate sample wells, and 2 xcexcL of DMSO was added to NSB and total binding (TB) wells. Finally, 25 xcexcL of membrane was added to each well.
The plates were covered and incubated at 37xc2x0 C. for 2 hours in a humidified incubator. Wells were aspirated on a Millipore vacuum manifold, and the wells were washed with 150 xcexcL isotonic saline solution. Wells were again aspirated. The plates were then dried for 1 hour in an 80xc2x0 C. vacuum drying oven. Plates were placed on a Millipore filter punch apparatus, and filters are placed in 12xc3x9775 mm polypropylene culture tubes. The samples were counted on a Packard gamma counter.
Using 125I- Echistatin (specific activity=2000 Ci/mmol) supplied by Amersham at a final concentration of 50 pM, the following parameters were routinely observed;
Input 80000 cpm
Total Counts 8000 cpm
Non-specific binding 200 cpm
Analysis of Results
The individual well activity was expressed as a percentage of the specific binding; % Max, and reported as the meanxc2x1standard deviation. Dose-inhibition relationships were generated for dose (X-axis) vs. % Max (Y-axis) for active compounds using a non-linear regression computer program (PS-NONLIN), and IC50 values with corresponding 95% confidence intervals were estimated from 50% of maximal attachment.
Reference Compounds:
Various Arginine-Glycine-Aspartic Acid (RGD)-containing peptides were assessed for the ability to inhibit xcex1vxcex23 binding and the corresponding IC50 values with 95% confidence intervals were generated; peptide structures are given by the standard single letter designation for amino acids. Values obtained compared favorably with adhesion assay results.
1. Nesbitt, S. A. And M. A. Horton, (1992), A nonradioactive biochemical characterization of membrane proteins using enhanced chemiluminescence, Anal. Biochem., 206 (2), 267-72.
Measuring the effect of compounds on the RGD-dependent attachment of cells to osteopontin mediated by the xcex1vxcex23 integrin.
Reagents
Cell Suspension Media: The cells were suspended for assay in the tissue culture media used for normal culture maintenance buffered with 25 mM HEPES (pH 7.4) without serum supplementation.
Compound Dilution Media: The stock compounds were dissolved in an appropriate vehicle (typically DMSO) and subsequently diluted in the tissue culture media used for normal culture maintenance buffered with 25 mM HEPES (pH 7.4) supplemented with 0.2% BSA (no serum); final vehicle concentration is xe2x89xa60.5%.
Plate Preparation
Human recombinant osteopontin (prepared as described in Stubbs, J. III, Connective Tissue Research, (1996) 35, (1-4), 393-399) was diluted to an appropriate concentration in Dulbecco""s phosphate buffered saline (D-PBS) without calcium or magnesium, pH 7.1. 100 xcexcL of this solution was incubated in the wells of PRO-BIND assay plates (Falcon 3915) for 2 hours at 37xc2x0 C. Following incubation the wells were aspirated and washed once with D-PBS; plates can either be used immediately or stored for up to 1 week at 4xc2x0 C. Prior to assay, the wells were blocked with 1% bovine serum albumin (BSA) in cell suspension media for 1 hour at 37xc2x0 C. Following the blocking period, wells were aspirated and washed once with D-PBS.
Cell Suspension
xcex1vxcex23-expressing cell lines are maintained by standard tissue culture techniques. For assay, the cell monolayer was washed three times with D-PBS, and the cells were harvested with 0.05% trypsin/0.53 mM EDTA (GIBCO). The cells were pelleted by low-speed centrifugation and washed three times with 0.5 mg/mL trypsin inhibitor in D-PBS (Sigma). The final cell pellet was resuspended in cell suspension media at a concentration of 106 cells/mL.
Attachment Assay
Incubation: 100 xcexcL of diluted test compound was added to osteopontin-coated wells (in triplicate) followed by 100 xcexcL of cell suspension; background cell attachment was determined in uncoated wells. The plate was incubated at 25xc2x0 C. in a humidified air atmosphere for 1.5 hours. Following the incubation period, the wells were gently aspirated and washed once with D-PBS.
Cell Number Detection: The number of cells attached was determined by an MFT dye conversion assay (Promega) according to the manufacturer""s instructions. Briefly, MTT dye was diluted in cell suspension media (15:85) and 100 xcexcL was added to each well. The assay plates were incubated for 4 hours at 37xc2x0 C. in a humidified 5% CO2/95% air atmosphere, followed by the addition of 100 xcexcL stopping/solubilization solution. The assay plates were covered and incubated at 37xc2x0 C. in a humidified air atmosphere overnight. After the solubilization period, the optical density of the wells was measured at a test wavelength of 570 nM with a reference measurement taken simultaneously at 630 nM.
Analysis of Results
The individual well optical density was expressed as a percentage of the maximal attachment (% Max) wells minus background attachment, and reported as the meanxc2x1standard deviation. Dose-inhibition relationships were generated for dose (X-axis) vs. % Max (Y-axis) for active compounds using a non-linear regression computer program (PS-NONLIN), and IC50 values with corresponding 95% confidence intervals were estimated from 50% of maximal attachment. Reference Compounds
Various Arginine-Glycine-Aspartic Acid (RGD)-containing peptides, and monoclonal antibodies were assessed for the ability to inhibit osteopontin-xcex1vxcex23 attachment and the corresponding IC50 values with 95% confidence intervals were generated in the SK-MEL-24 human malignant melanoma cell line; peptide structures are given by the standard single letter designation for amino acids:
Ruoslahti, R. Fibronectin and its receptors. Ann. Rev. Biochem. 57:375-413, 1988.
Hynes, R. O. Integrins: Versatility, modulation, and signaling in cell adhesion. Cell. 69:11-25,1992.
The results of this test procedure on representative compounds of this invention are shown in Table I.
Osteoclast Bone Pitting
The test procedure was conducted as described by R. J. Murrills and D. W. Dempster, Bone, 11, 333-344(1990). Briefly, 4xc3x974xc3x970.2 mm slices of devitalized bovine cortical bone were numbered, placed in the wells of 96-well culture plates and wetted with 100 ul of Medium 199 containing Hanks salts, 10 mM HEPES, pH 7.0 (Medium 199/Hanks). Bone cell suspensions containing osteoclasts were prepared by mincing the long bones of neonatal rats (Sprague-Dawley, 4-6 days old) in Medium 199/Hanks. 100 uL of the suspension were then plated onto each slice and incubated 30 minutes to allow osteoclasts to adhere. The slices were rinsed to remove non-adherent cells and incubated 24 h in Medium 199 containing Earle""s salts, 10 mM HEPES and 0.7 g/L NaHCO3, which equilibrates at pH 6.9 in a 5% CO2 atmosphere. At this pH the adherent osteoclasts excavate an adequate number of resorption pits for assay purposes. Slices were fixed in 2.5% glutaraldehyde and osteoclasts counted following tartrate-resistant acid phosphatase staining. In experiments in which osteoclast numbers were significantly reduced in a particular treatment, a check is made for non-specific cytotoxicity by counting the number of contaminant fibroblast-like cells following toluidine staining. All cells were stripped from the slice by sonication on 0.25M NH4OH and the resorption pits formed by the osteoclasts during the experiment stained with toluidine blue. Resorption pits were quantified by manually counting.
Statistics
The experiments were conducted according to a block design with osteoclasts from each animal exposed to each treatment. Three replicate slices were used per treatment per animal, such that a total of 96 slices were examined for an experiment involving four animals and eight treatments (including control). Several parameters were recorded on a xe2x80x9cper slicexe2x80x9d basis: number of pits, number of osteoclasts, number of pits per osteoclast, number of fibroblast-like bone cells. SAS or JMP statistical software were used for statistical analysis. If analysis of variance reveals significant effects in the experiment, those treatments differing significantly from control were identified using Dunnett""s test. IC50s were calculated using dose-response curves.
Reference Compound: Rat Calcitonin
Clinical Relevance
Osteoclasts are responsible for the bone loss that occurs at the onset of osteoporosis and anti-resorptive drugs directed against the osteoclast are a requirement for patients losing bone. Calcitonin and bisphosphonates, both used as anti-resorptives in the clinic, show significant osteoclast inhibitory activity in this test procedure. Hence it is a reasonable test procedure in which to identify novel anti-resorptives.
The results of this test procedure on representative compounds of this invention is shown in Table II.
Effects of Test Compounds on PTH-induced Hypercalcemia of Thyro-parahyroidectomized Male Rats.
Male thyro-parathyroidectomized (TPTX) rats (Charles River) were randomly assigned to groups of 7 rats/group. Following a baseline serum calcium determination an Alzet 1003D minipump (Alza Corporation, Palo Alto, Calif.) loaded with 0.3 mg/ml PTH (Bachem, Philadelphia, Pa.) was implanted subcutaneously in each rat. For evaluation of prophylactic effects of a test drug, another minipump with appropriate concentration of the test drug solution was implanted subcutaneously at a site away from PTH minipump or implanted as a pellet of the test compound away from the PTH minipump. Alternatively, test drugs were administered by oral gavage as a solution or uniform suspension in an appropriate medium depending on the physical properties of the test compound. A group of 7 unimplanted TPTX rats was set aside as a normal control group. Twenty hours after minipump implantation blood was collected from each rat to confirm the presence of hypercalcemia (udged by elevation of serum calcium levels, 2 SD greater than normal non-implanted level). At various intervals between 0.5 and 24 hours after dosing (usually one to three time points), blood was collected from each rat and the serum evaluated for total calcium. Serum calcium levels were measured using the Nova 7+7 calcium auto analyzer spectrophotometrically using the Sigma test kit (#587A). Test results were determined by the difference in serum calcium between vehicle and treatment group following PTH administration, using a oneway analysis of variance with Dunnett""s test or other multiple comparison methods and are displayed in Tables III-V.
1. Takeuchi M, Sakamoto S, Kawamuki K, Kudo M, Abe T, Fujita S, Murase K, and Isomura Y, (1990). Synthesis and structure activity relationship of new bisphosphonate derivative. Abstract #53, 199th American Chemical Society Meeting, Boston, Mass.
2. Fisher J, Caulfield M, Sato M, Quartuccio H, Gould R, Garsky V, Rodan G, Rosenblatt M, (1993). Inhibition of osteoclastic bone resorption in vivo by echistatin, an xe2x80x9carginyl-glycyl-aspartylxe2x80x9d (RGD)-containing protein. Endocrinology, Vol. 132 (3) 1411-1413.
Measuring the effect of compounds on ADP-induced platelet aggregation mediated by a fibrinogen-xcex1IIbxcex23 integrin interaction.
Test Procedure
Human Platelets: Platelet-enriched plasma was obtained commercially from a donor pool. The plasma was tested prior to shipment and found to be negative for HIV, HCV and Hepatitis B. Platelet-rich plasma (PRP) was obtained by diluting plasma to an approximate final concentration of 3xc3x9710(6) platelets per mL in platelet poor plasma (PPP). PPP was the supernatant of a lowspeed centrifugation of plasma.
Adenosine diphosphate (ADP): ADP was obtained commercially and diluted to 1 mM (stock solution) in distilled, deionized water (ddH2O).
Platelet Aggregation
Incubation: PRP and PPP were prewarmed in a water bath at 37xc2x0 C. The sample compounds were dissolved in an appropriate vehicle (typically DMSO) and diluted in vehicle to 100xc3x97 of the testing concentration. PRP plus sample compound in a final volume of 500 uL was added to a pre-warmed cuvette in a ChronoLog aggregometer. A control containing PRP and 5 uL of vehicle was treated similarly to the test cuvette; final vehicle concentration was 1%. The two cuvettes were incubated with stirring (1000 rpm) at 37xc2x0 C. for 5 minutes. Five hundred microliters of PPP was used as a reference (100% aggregation).
Aggregation: To begin the test, ADP was added yielding a final concentration of 20 uM to both samples (plus and minus sample compound). Light transmittance was monitored continuously and compared to the reference cuvette. After five minutes, the test was terminated and the slope and maximal amplitude of the resulting aggregation plot was calculated by the aggregometer.
Analysis of Results
The percent of maximal aggregation is the ratio of the maximal aggregations of the sample cuvette to the control multiplied by 100 (% Max) and reported as the mean+xe2x88x92standard deviation. Dose-inhibition relationships were generated for dose (X-axis) vs. % Max (Y-axis) for active compounds using a non-linear regression computer program (PS-NONLIN) and ICSO values with corresponding 95% confidence intervals were estimated from 50% of maximal aggregation.
Reference Compounds
Known Arginine-Glycine-Aspartic Acid (RGD)-containing peptides, and snake venoms were tested for their ability to inhibit ADP induced platelet aggregation; peptide structures are given by the standard single letter designation for amino acids. Results are shown in Table VI.
Foster M., Hornby E., Brown S., Kitchin J., Hann M. and P. Ward. Improved Potency and Specificity of ARG-GLYASP (RGD) Containing Peptides as Fibrinogen Receptor Blocking Drugs. Thromb Res 1993; 72:231-245.
Ramjit D., Lynch J., Sitko G., Mellott J., Holahan M., Stabilito I., Stranierie M., Zhang G., Lynch R., Manno P., Chang C., Nutt R., Brady S., Veber D., Anderson P., Shebuski R., Friedman P. and R. Gould. Antithrombotic Effects of MK-0852, a Platelet Fibrinogen Receptor Antagonist, in Canine Models of Thrombosis. J. Pharmacol Exp.Ther 1993; 266(3):1501-1511.
Platelet aggregation Test Results for sample compounds are displayed in Table VII.
When the compounds are employed for the above utilities, they may be combined with one or more pharmaceutically acceptable carriers, for example, solvents, diluents and the like, and may be administered orally in such forms as tablets, capsules, dispersible powders, granules, or suspensions containing, for example, from about 0.05 to 5% of suspending agent, syrups containing, for example, from about 10 to 50% of sugar, and elixirs containing, for example, from about 20 to 50% ethanol, and the like, or parenterally in the form of sterile injectable solutions or suspensions containing from about 0.05 to 5% suspending agent in an isotonic medium. Such pharmaceutical preparations may contain, for example, from about 25 to about 90% of the active ingredient in combination with the carrier, more usually between about 5% and 60% by weight.
The effective dosage of active ingredient employed may vary depending on the particular compound employed, the mode of administration and the severity of the condition being treated. However, in general, satisfactory results are obtained when the compounds of the invention are administered at a daily dosage of from about 0.5 to about 500 mg/kg of animal body weight, preferably given in divided doses two to four times a day, or in a sustained release form. For most large mammals the total daily dosage is from about 1 to 100 mg, preferably from about 2 to 80 mg. Dosage forms suitable for internal use comprise from about 0.5 to 500 mg of the active compound in intimate admixture with a solid or liquid pharmaceutically acceptable carrier. This dosage regimen may be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
These active compounds may be administered orally as well as by intravenous, intramuscular, or subcutaneous routes. Solid carriers include starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and kaolin, while liquid carriers include sterile water, polyethylene glycols, non-ionic surfactants and edible oils such as corn, peanut and sesame oils, as are appropriate to the nature of the active ingredient and the particular form of administration desired. Adjuvants customarily employed in the preparation of pharmaceutical compositions may be advantageously included, such as flavoring agents, coloring agents, preserving agents, and antioxidants, for example, vitamin E, ascorbic acid, BHT and BHA.
The preferred pharmaceutical compositions from the standpoint of ease of preparation and administration are solid compositions, particularly tablets and hard-filled or liquid-filled capsules. Oral administration of the compounds is preferred.
These active compounds may also be administered parenterally or intraperitoneally. Solutions or suspensions of these active compounds as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid, polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exits. It must be stable under conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oil.
The compounds of Formulae (I) and (II) of this invention are useful in treating conditions in mammals characterized by bone resorption of mineralized tissue such as in osteoporosis, hypercalcemia of malignancy, osteopenia due to bone metastases, periodontal disease, hyperparathyroidism, periarticular erosions in rheumatoid arthritis, Paget""s disease, immobilization-induced osteopenia or glucocorticoid treatment.
In particular, compounds of Formulae (I) and (II) of this invention are therapeutically useful in the treatment and/or prevention of osteoporosis in mammals.
The compounds of this invention and their preparation can be understood further by the following examples, but should not constitute a limitation thereof.